Method for treating cancer

ABSTRACT

The present disclosure relates to a method of treating prostate cancer comprising administering to a subject in need thereof a therapeutically effective amount of a vasopressin analogue.

RELATED APPLICATIONS

This application claims priority to U.S. 62/139,976, filed Mar. 30,2015, the entire contents of which is hereby incorporated by reference.

FIELD

The present disclosure relates to a method of treating cancer using avasopressin analogue. In particular, the disclosure relates to a methodof treating prostate cancer comprising administering a vasopressinanalogue.

INTRODUCTION

Prostate cancer is the second leading cause of cancer-related deaths inWestern countries.¹ Most patients dying of prostate cancer havemetastatic castrate resistant disease (CRPC).² There is no curativeagent for treatment of CRPC.

Docetaxel is first drug of choice for management of CRPC.^(3,4) AlthoughDocetaxel-based combination chemotherapy has significantly improvedsurvival of CRPC patients, durable responses are uncommon.⁴⁻⁶Furthermore, Docetaxel causes adverse events such as grade 3 or 4neutropenia, fatigue, alopecia and nausea. High dose docetaxel inducessignificant toxicity.^(4,5,7)

Desmopressin is a synthetic derivative of antidiuretic hormone.Desmopressin is a safe and effective hemostatic agent in patients withvon Willbrand disease, hemophilia A and other bleeding disorders.⁸⁻¹⁰Desmopressin induces an increase in the plasma level of coagulationfactor VIII, von Willbrand factor (VWF) and tissue plasminogen activator(t-PA).⁹ Recent reports suggest that desmopressin inhibits tumormetastasis in in vivo models.¹¹⁻¹³ Alonso et al. reported thatdesmopressin inhibits lung colonization by blood-borne breast cancercells in an in vivo model.¹¹ Desmopressin injected preoperativelyreduced lymph node and lung metastasis in a mammary tumor model.¹²Desmopressin impairs aggressiveness of residual mammary tumors duringchemotherapy.¹³

SUMMARY

The present disclosure relates to a method of treating cancer using avasopressin analogue, such as desmopressin. In particular, the methodrelates to the treatment of prostate cancer.

In one embodiment, the disclosure includes a method of treating prostatecancer comprising administering to a patient in need thereof atherapeutically effective amount of desmopressin or a vasopressinanalogue thereof.

In one embodiment, the method further comprises co-administering to apatient in need thereof a pharmaceutical composition a taxane or afunctional derivative thereof.

In one embodiment, the disclosure relates to a method of treatingmetastatic castrate-resistant prostate cancer, comprising administeringto a subject in need thereof, a therapeutically effective amount ofvasopressin analogue, such as desmopressin.

In another embodiment of the disclosure, there is also included a use ofa therapeutically effective amount of a vasopressin analogue, such asdesmopressin, for the treatment of prostate cancer. In one embodiment,the disclosure includes a use of a therapeutically effective amount of(i) desmopressin or a vasopressin analogue thereof; and (ii) a taxane ora functional derivative thereof, for the treatment of prostate cancer.

In another embodiment of the disclosure, there is also included a use ofa therapeutically effective amount of a vasopressin analogue, such asdesmopressin, for the treatment of metastatic castrate-resistantprostate cancer.

The disclosure also includes kit for the treatment of prostate cancer,comprising (i) a therapeutically effective amount of a vasopressinanalogue, such as desmopressin, and (ii) a therapeutically effect amountof a taxane or a functional derivative thereof, and instructions forusing the kit.

Other features and advantages of the present application will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the application aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the application will becomeapparent to those skilled in the art from this detailed description.

DRAWINGS

The disclosure will now be described in greater detail with reference tothe following drawings in which

FIG. 1A is a bar graph showing the effects of desmopressin on PC3 cells;FIG. 1B is a bar graph showing the effects of docetaxel on PC3 cells;FIG. 1C is a bar graph showing the effects of docetaxel anddesmopressin, alone or in combination, on PC3 cells;

FIG. 2A are micrographs showing the effect of docetaxel anddesmopressin, alone or in combination, on cell migration; FIG. 2B aremicrographs showing the effect of docetaxel and desmopressin, alone orin combination, on cell invasion; FIG. 2C is a bar graph showing theeffect of docetaxel and desmopressin, alone or in combination, on cellmigration;

FIG. 3A are micrographs showing the anti-invasive effects of docetaxeland desmopressin, alone or in combination; FIG. 3B is a bar graphshowing the anti-invasive effects of docetaxel and desmopressin, aloneor in combination;

FIG. 4A shows a Western blot analysis on cell proliferation effects ofdocetaxel and desmopressin, alone or in combination; FIG. 4B shows aWestern blot analysis on cell apoptosis effects of docetaxel anddesmopressin, alone or in combination; FIG. 4C is a bar graph showingthe effects of docetaxel and desmopressin, alone or in combination, onthe ratio of Bax/Bcl-2; FIG. 4D is a bar graph showing the cell cycleeffects of docetaxel and desmopressin, alone or in combination;

FIGS. 5A-5D are bar graphs showing the effects of desmopressin onexpression of pro-uPA, active uPA, MMP-2, MMP-9, and uPAR;

FIGS. 6A-6D are bar graphs showing the effects of desmopressin in atime-dependent manner on the expression of pro-uPA, active uPA, MMP-2,MMP-9, and uPAR;

FIG. 7 is a schematic diagram showing, in one embodiment, the effects ofdesmopressin on tumour cell migration and invasion via the uPA-MMPpathway;

FIGS. 8A-8D are bar graphs showing the effects of docetaxel anddesmopressin, alone or in combination, on expression of pro-uPA, activeuPA, MMP-2, MMP-9, and uPAR;

FIG. 9 is a graph showing the effects of docetaxel and desmopressin,alone or in combination, on tumour volume, and also showing thetreatment schedule;

FIG. 10 is a bar graph showing the effects of docetaxel (DTX) ordesmopressin (Desmo), alone and/or in combination, on DU145 cells;

FIG. 11 is a bar graph showing the effects of docetaxel (DTX) ordesmopressin (Desmo), alone and/or in combination, on DU145 cells,assessed using wound healing assay;

FIG. 12A-D are micrographs showing the effects of docetaxel (DTX) ordesmopressin (Desmo), alone and/or in combination, on DU145 cells,assessed using wound healing assay;

FIG. 13 is a graph depicting the effects of docetaxel and desmopressin,alone and/or in combination, on tumor volume in a prostate cancerxenograft model;

FIG. 14 is a graph depicting the effects of docetaxel and desmopressin,alone and/or in combination, on body weight of animals in a prostatecancer xenograft model;

FIG. 15A-D are micrographs showing the effects of docetaxel anddesmopressin, alone and/or in combination, on tumor size in a prostatecancer xenograft model.

DESCRIPTION OF VARIOUS EMBODIMENTS (I) Definitions

The term “vasopressin analogue” as used herein, refers to compounds orderivatives having similar function to vasopressin but not necessarily asimilar structure, and includes all compounds or derivatives havinganti-proliferative activity, including prodrugs. Vasopressin analoguesinclude, but are not limited to, synthetic arginine vasopressin, lysinevasopressin, terlpressin, felypressin, or omipressin. As used herein,the term “desmopressin” or “DDAVP®”, a vasopressin analogue, refers to1-desamino-8-D-arginine vasopressin.

As used herein, the term “taxane” generally refers to a class ofditerpenes produced and isolated from natural sources such as the plantsof the genus Taxus (Yew tree), or from cell culture. This term alsoincludes those taxanes that have been artificially synthesized. Forexample, this term includes docetaxel and paclitaxel, and derivativesthereof. Also included are “functional derivatives” of taxanes alsohaving anti-proliferative activity, including prodrugs.

The term “pharmaceutically acceptable” as used herein means compatiblewith the treatment of subjects, for example, humans.

The term “pharmaceutically acceptable salt” refers, for example, to asalt that retains the desired biological activity of a compound of thepresent disclosure and does not impart undesired toxicological effectsthereto; and may refer to an acid addition salt or a base addition salt.

As used herein, the phrase “castrate resistant prostate cancer” (alsoknown as hormone-refractory prostate cancer or androgen-independentprostate cancer or endocrine resistant prostate cancer) refers toprostate cancer which is resistant to hormone therapy.

As used herein, the term “metastatic” is defined as the transfer ofcancer cells from one organ or part to another not directly connectedwith it.

As used herein, a “subject” refers to all members of the animal kingdomincluding mammals, and suitably refers to humans. A member of the animalkingdom includes, without limitation, a mammal (such as a human,primate, swine, sheep, cow, equine, horse, camel, canine, dog, feline,cat, tiger, leopard, house pet, livestock, rabbit, mouse, rat, guineapig or other rodent, seal, whale and the like). In an embodiment of thepresent disclosure, the subject is in need of a treatment of thedisclosure.

As used herein, the term “prodrug” refers to a substance that isprepared in an inactive form that is converted to an active form (i.e.,drug) within the body or cells thereof by the action of, for example,endogenous enzymes or other chemicals and/or conditions. Prodrugderivatives of desmopressin, or pharmaceutically acceptable salts orsolvates thereof, can be prepared by methods known to those of ordinaryskill in the art.

The term “therapeutically effective amount” as used herein means anamount effective, at dosages and for periods of time necessary toachieve the desired result. Effective amounts may vary according tofactors such as the disease state, age, sex and/or weight of thesubject. The amount of a given compound or composition that willcorrespond to such an amount will vary depending upon various factors,such as the given drug or compound, the pharmaceutical formulation, theroute of administration, the identity of the subject being treated, andthe like, but can nevertheless be routinely determined by one skilled inthe art.

The term “administered” or “administering” as used herein meansadministration of a therapeutically effective dose of a compound orcomposition of the disclosure to a subject.

In understanding the scope of the present disclosure, the term“comprising” and its derivatives, as used herein, are intended to beopen ended terms that specify the presence of the stated features,elements, components, groups, integers, and/or steps, but do not excludethe presence of other unstated features, elements, components, groups,integers and/or steps. The foregoing also applies to words havingsimilar meanings such as the terms, “including”, “having” and theirderivatives. Finally, terms of degree such as “substantially”, “about”and “approximately” as used herein mean a reasonable amount of deviationof the modified term such that the end result is not significantlychanged. These terms of degree should be construed as including adeviation of at least ±5% of the modified term if this deviation wouldnot negate the meaning of the word it modifies.

(II) Method for the Treatment of Cancer

The present disclosure relates to the treatment of cancer. Inparticular, the present disclosure relates to the treatment of prostatecancer, comprising administering to a patient in need thereof atherapeutically effective amount of a vasopressin analogue. In otherembodiments, the disclosure includes a method for the treatment ofhormone dependent cancers, such as lung, uterine, kidney, ovarian,testicular, breast or colorectal comprising administering to a patientin need thereof a therapeutically effective amount of a vasopressinanalogue.

In one embodiment, the present disclosure relates to a method oftreating prostate cancer comprising administering to a patient in needthereof a therapeutically effective amount of a vasopressin analogue. Inone embodiment, the vasopressin analogue is desmopressin, syntheticarginine vasopressin, lysine vasopressin, terlipressin, felypressin, oromipressin. In a further embodiment, the vasopressin analogue isdesmopressin.

In another embodiment of the disclosure, there is also included a use ofa therapeutically effective amount of a vasopressin analogue, for thetreatment of prostate cancer. In another embodiment of the disclosure,there is also included a use of a therapeutically effective amount of avasopressin analogue, for the manufacture of a medicament for thetreatment of prostate cancer. In one embodiment, the vasopressinanalogue is desmopressin. In other embodiments, the disclosure includesa use of a therapeutically effective amount of a vasopressin analogue,for the treatment of hormone dependent cancers, such as lung, uterine,kidney, ovarian, testicular, breast or colorectal.

In one embodiment, the prostate cancer is metastatic prostate cancer. Inanother embodiment, the prostate cancer is castrate-resistance prostatecancer. In a further embodiment, the cancer is metastaticcastrate-resistant prostate cancer.

In another embodiment of the disclosure, desmopressin or an analoguethereof is administered to the subject, for example, a prodrug ofdesmopressin. Prodrugs of desmopressin include aliphatic carboxylic acidesters and carbonate esters of the tyrosine phenolic group. In anotherembodiment, desmopressin is administered to the subject.

In another embodiment of the disclosure, the method further comprisesco-administering a taxane. As used herein, the term “co-administer,” isintended to embrace separate administration of the vasopressin analogue,such as desmopressin, and the taxane (or functional derivative) in asequential manner as well as co-administration of these agents in asubstantially simultaneous manner, such as in a singlemixture/composition or in doses given separately, but nonethelessadministered substantially simultaneously to the subject.

In one embodiment, the taxane is docetaxel, paclitaxel, larotaxel,cabazitaxel, baccatin, cephalomannine, brevifoliol, BMS-275183,abraxane, taxoprexin, xytotax or functional derivatives thereof. Inanother embodiment, the taxane is docetaxel, paclitaxel, cabazitaxel orfunctional derivatives thereof. In a further embodiment, the taxane isdocetaxel.

In an embodiment of the disclosure, the vasopressin analogue isadministered at a dose effective to reduce or halt proliferation of thecancer cells, for example by inducing cell cycle arrest. In oneembodiment, the cancer cells are prostate cancer cells. In anotherembodiment, the vasopressin analogue is desmopressin.

In another embodiment of the disclosure, the vasopressin analogue isadministered at a dose effective to reduce or halt cancerous tumourgrowth, and decrease tumour volume. In one embodiment, the canceroustumour growth is a cancerous prostate tumour. In one embodiment, thevasopressin analogue is desmopressin.

In another embodiment of the disclosure, the vasopressin analogue isadministered at a dose effect to reduce or prevent metastases of cancercells to other tissues and organs of the subject's body. In oneembodiment, the cancer cells are prostate cancer cells. In oneembodiment, the vasopressin analogue is desmopressin.

In another embodiment of the disclosure, the vasopressin analogue isco-administered with a taxane or functional derivative thereof, at adose which enhances the anti-proliferative efficacy of the taxane. Inone embodiment, the vasopressin analogue is desmopressin. It is wellknown to those skilled in the art that taxanes cause seriousside-effects such as grade 3 or 4 neutropenia. The administration of avasopressin analogue, such as desmopressin, thereof allows for the useof less toxic doses of the taxane without compromising efficacy of thetaxane for the treatment of cancer.

In another embodiment of the disclosure, there is included a kit for thetreatment of cancer. In one embodiment, the cancer is prostate cancer.

In one embodiment, the disclosure includes a kit for the treatment ofcancer, comprising (i) a therapeutically effective amount of avasopressin analogue, and (ii) a therapeutically effect amount of ataxane or a functional derivative thereof; and instructions for usingthe kit. In one embodiment, the vasopressin analogue is desmopressin.

In one embodiment, the kit is for the treatment of prostate cancer,lung, uterine, kidney, ovarian, testicular, breast or colorectal. In oneembodiment, the cancer is prostate cancer. In another embodiment, theprostate cancer is metastatic prostate cancer. In another embodiment,the kit is for the treatment of castrate-resistance prostate cancer. Ina further embodiment, the kit is for the treatment of metastaticcastrate-resistant prostate cancer.

In another embodiment of the disclosure, the kit comprises a vasopressinanalogue which is administered to the subject. In another embodiment,the kit comprises desmopressin which is administered to the subject.

In another embodiment of the disclosure, the kit comprises a taxanewhich is co-administered (with the vasopressin analogue) to the subject.Co-administration encompasses separate administration of the vasopressinanalogue (such as desmopressin) and the taxane (or functionalderivative) in a sequential manner as well as co-administration of theseagents in a substantially simultaneous manner, such as in a singlemixture/composition or in doses given separately, but nonethelessadministered substantially simultaneously to the subject.

In one embodiment, the kit comprises a taxane which is docetaxel,paclitaxel, larotaxel, cabazitaxel, baccatin, cephalomannine,brevifoliol, BMS-275183, abraxane, taxoprexin or xytotax, or functionalderivatives thereof. In another embodiment, the taxane is docetaxel,paclitaxel, cabazitaxel or functional derivatives thereof. In a furtherembodiment, the taxane is docetaxel.

In one embodiment, the vasopressin analogue, such as desmopressin, isformulated as a pharmaceutically acceptable salt, such as desmopressinacetate. In a further embodiment, the vasopressin analogue is formulatedin a pharmaceutical composition, comprising the vasopressin analogue anda pharmaceutically acceptable excipient and/or carrier. In oneembodiment, the vasopressin analogue is desmopressin.

In one embodiment, the vasopressin analogue, for example desmopressinacetate, is formulated as an aqueous solution of desmopressin acetatepresent at a concentration of about 4 μg/ml or about 15 μg/ml. In oneembodiment, the vasopressin analogue is administered at a dose ofbetween about 0.1 μg/kg to about 1.0 μg/kg, optionally 0.2 μg/kg toabout 0.5 μg/kg or about 0.4 μg/kg. In one embodiment, the vasopressinanalogue is formulated for intravenous, intramuscular or subcutaneousadministration. In one embodiment, the vasopressin analogue isformulated for immediate release, IV infusion, delayed release or depotadministration.

In one embodiment, the taxane or functional derivative thereof isformulated in a pharmaceutical composition, comprising the taxane and apharmaceutically acceptable excipient and/or carrier.

In one embodiment, the taxane is docetaxel. In a further embodiment, thedocetaxel is formulated in an aqueous solution present at aconcentration of between about 50 mg/ml to about 100 mg/ml, optionallyabout 80 mg/ml. In one embodiment, the docetaxel is administered at adose between about 50 mg/m2 to about 100 mg/m2, or about 75 mg/m2, every3 weeks as a 1 hour intravenous infusion. Optionally, prednisone 5 mgorally twice daily is administered concurrently with the docetaxel. Inone embodiment, the docetaxel is formulated for intravenousadministration.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersion and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists.

In one embodiment, the taxane is administered to the patient every threeweeks for four cycles, and the vasopressin analogue is administeredabout 30 minutes before the dose of taxane is administered, and also 24hours after the dose of taxane is administered.

Although the disclosure has been described in conjunction with specificembodiments thereof, if is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. In addition, citation or identification of anyreference in this application shall not be construed as an admissionthat such reference is available as prior art to the present disclosure.

The operation of the disclosure is illustrated by the followingrepresentative examples. As is apparent to those skilled in the art,many of the details of the examples may be changed while stillpracticing the disclosure described herein.

(III) Examples

The operation of the disclosure is illustrated by the followingrepresentative examples. As is apparent to those skilled in the art,many of the details of the examples may be changed while stillpracticing the disclosure described herein.

Example I

Materials and Methods

Cell Culture

PC3 human prostate carcinoma cells were obtained from the American TypeCulture Collection (Rockville, Md., USA). Cells were cultured inDMEM/F12 medium (Invitrogen, ON, Canada) with 10% fetal bovine serum(FBS; Gibco, NY, USA), 100 IU/m penicillin, 100 μg/ml streptomycin and0.3 mg/ml 1-glutamine, and maintained at 37° C. in a humidifiedatmosphere of 5% CO₂ in air. Cells were grown to 80% confluence in 10-cmtissue culture plates.

Chemicals

Docetaxel was purchased from Santa Cruz Biotechnology (CA, USA).Docetaxel were prepared in dimethyl sulfoxide (DMSO; Sigma, St Louis,USA) and diluted with cell culture medium at a final concentration of0.01% DMSO. Desmopressin was kindly provided to us by FerringPharmaceutical (CA, USA).

Cell Proliferation Assay

Cell proliferation was determined by MTS assay as previouslydescribed.¹⁴ Cells (5×10³/well) were plated using 96 well plates. Aftercultivation for 24 h, a range of concentration of desmopressin (100 pM-1μM) and/or Docetaxel (1 nM-100 nM) were added and culture continued forup to 72 h. Cell viability was assessed by incubation with MTS for 2hours and resulting absorbance measured at 595 nm using an ELISA platereader. In the combination studies, varying dose of each agents weretested. The experiments were repeated 3 times in triplicates andstatistical analysis performed.

Wound Healing Assay

Cell motility was assessed using a wound healing assay performedaccording to a protocol described by Liang et al.¹⁵. Cells (2×10⁴/well)were plated in 6-well plate. Cells at over 90% confluence were incubatedfor 1 hr with 1 mg/l of mitomycin C (Sigma-Aldrich, St Louis, USA) inorder to exclude proliferative effect. After mitomycin C treatment, theinjury line was made using a 200 μl tip, and the cell monolayers wererinsed with PBS. The cells were treated with desmopressin and/ordocetaxel and allowed to migrate for 24 h. A computer-based microscopyimaging system was used to determine wound healing after scratching theplate with a microscope at 200× magnification. Several wound areas wereobserved for cell migration. The experiments were repeated 3 times intriplicates.

Cell Migration and Invasion Assay

The migration of cells was measured using transwell insert plates (BDBiosciences, Bedford Mass.) according to the manufacture's protocol.Cells were subjected to 24 h of serum deprivation in DMEM/F12supplemented with FBS. PC3 (5×10⁴) cells were plated onto filters in8.0-μm transwell insert plates and treated with these compounds,desmopressin (1 nM, 1 μM) and 5 nM docetaxel, in serum-free medium. Thelower chamber also contained 10% FBS. Cells were allowed to migrate for24 h. After the treatment, cells remaining on the top surfaces of thefilter were removed using cotton swabs. The migrated cells that areattached to the lower surface of the filter were fixed in 4%formaldehyde at room temperature for 30 min, and then stained withcrystal violet for 20 min. The migrated cells from random fields werechosen and counted using the computer-based microscopy imaging system.For the invasion assay, the same procedures were performed as describedin the migration assay, except that the cells were plated onto 24-wellmatrigel-coated transwell plates (BD Biosciences, Bedford Mass.). Theexperiments were repeated 2 times in duplicates and statistical analysisperformed.

Flow Cytometry

To analyze cell cycle profiles, cells were plated at a density of 1×10⁶per 10 cm dish. Asynchronously growing cells were pulse labeled with 10mM bromodeoxyuridine (BrdU) for 2 h with or without prior treatment ofthe antioxidants at the end of 24 h. Cells were then harvested, fixedwith 70% ethanol, treated with 0.1% HCl and heated for 10 min at 90° C.to expose the labeled DNA. Cells were stained with anti-BrdU-conjugatedFITC (Becton-Dickinson) and counterstained with propidium iodide, andthen allowed to incubate for 30 mins on ice. Samples were filteredthrough a nylon mesh. Cell cycle analysis was carried out on the FACSCalibur flow cytometer using the Cell Quest Pro software package(Becton-Dickinson, Calif., USA). The experiments were repeated 3 timesin triplicates and statistical analysis performed as mentioned below.

Western Blot Analysis

Protein lysates from the desmopressin monotherapy dose response study(100 pM-1 uM), desmopressin monotherapy time point study (1 uM, 0-48 h)and combination study and concentrated media employed for Western blotanalysis, were prepared as previously described.¹⁴ The proteins weresubjected to 10-12% SDS-PAGE and electrophoretically transferred onto aPVDF membrane at 100V/300 mA overnight using a semidry transferapparatus (Bio-Rad Laboratories, Hercules, Calif.). After activation inmethanol (100%), blots were incubated for 60 mins at room temperature inTBST containing 5% skimmed milk. After washing, the membranes wereincubated with primary antibodies against Bax, bcl-2, p21 (waf1/cip1),p27 (kip1), cdk2, cdk4, uPA, uPAR, MMP-2 and MMP-9 (1:100-200, SantacruzBiotechnology, Santacruz, Calif., USA). After incubation with respectiveprimary antibody, the membrane was washed with TBST 3 times for 5 min,then incubated with appropriate secondary antibody for 1 h at roomtemperature and washed with TBST 3 times for 5 min. Protein detectionwas performed with enhanced chemiluminescence Western blotting regents(Amersham Pharmacia Biotech, Buckinghamshire, UK).

In Vitro Studies Using Xenograft

The mice were housed and maintained in laminar flow cabinets underspecific pathogen-free conditions in facilities approved by theUniversity of Toronto Animal Research Ethics Board and in accordancewith their regulations and standards by the Canadian Council on AnimalCare (CCAC). Cells (1×10⁵ PC3 cells with 100 μl martrigel solution (BDBiosciences, CA, USA)) were inoculated subcutaneously (sc) into 6-8week-old male nude mice (Harlan Sprague Dawley, Inc.). After 14 days,the developing tumors were measured and mice randomly assigned todifferent treatment groups. Tumor volumes were determined by measurementof tumor length (L) and width (W) with a caliper and calculatedaccording to the formula: V=(L×W²)(π/6) twice a week. A tumor volume inthe range of 100 mm³ was required for a given mouse to be included.Using the xenograft model, tumor growth was determined for the controland each treatment mice. Mice were randomized into four groups; control(n=15), desmopressin alone (n=15), docetaxel alone (n=10) anddesmopressin in combination with docetaxel (n=10). Control animalsreceived only the saline vehicle. Desmopressin from FerringPharmaceuticals (Ferring Inc, CA, USA) was administrated in 2 doses, 0 hand 24 h. Mice received desmopressin intravenously in the saline at afinal dose of 2 μg/mV body weight (50 ng/0.3 ml saline dose). Mice wereadministrated docetaxel at a dose of 5 mg/kg intravenously as 3 weeklycycles. Desmopressin was administrated in 2 doses, 30 min prior to and24 h after the administration of docetaxel. The animals that wereadministrated docetaxel or desmopressin in combination with docetaxelwere sacrificed 35 days after cell inoculation. Control and desmopressinmice were monitored until tumor volume was approximately 1500 mm3 or day49, whichever earlier, in order to evaluate antitumor effect ofdesmopressin in vivo study.

Statistical Analyses

All experiments were performed in triplicates. The date representedmean±the standard error of the mean. Statistical analysis was done byStudent's t test at a significance level of P<0.05. Analyses of the invivo results were performed using either Students t-testing or repeatedmeasures one-way ANOVA techniques.

Example 1—Effect of Docetaxel and/or Desmopressin on Cell Proliferationof PC3 Cells

Docetaxel and/or desmopressin inhibit cell proliferation on PC3 cells.The MTS cell proliferation assay was conducted to investigate whetherdocetaxel and/or desmopressin suppresses cell proliferation. The assaywas carried out at 72 h with the cells treated with a dose range of0-100 nM docetaxel. Cell growth was expressed as a relative value tothat of the untreated control cells. PC3 cells treated with docetaxelfor 72 h decreased cell viability in a dose dependent manner (FIG. 1A).Cells treated with a range of doses of desmopressin (1 nM-1 μM)significantly reduced cell proliferation compared to controls by 72 h(FIG. 1B, p<0.01). Moreover, combination therapy of desmopressin (1 nM,1 μM) and 5 nM docetaxel also resulted in a significant decrease in cellproliferation (FIG. 1C, p<0.01).

Example 2—Differential Influences of Desmopressin on Migration of PC3Cells in a Dose Dependent Manner

Since desmopressin and docetaxel treatment resulted inanti-proliferative effect on PC3 cells in culture, the effect ofdesmopressin alone (1 nM, 1 μM) and in combination with 5 nM docetaxeltreatment on cell migration by the wound-healing assay was determined.As shown in FIG. 2, the difference in distance migrated by controlbetween 0 h and 24 h was measured and compared with that of the treatedcells. At the end of 24 h, a significant decrease in cell migration witha combination of desmopressin and docetaxel was observed (FIG. 2A). Themigratory capacity of cells was further quantified using the migrationchamber transwell plates. Different doses of desmopressin (1 nM and 1μM) treatment significantly inhibited cell migration (FIGS. 2B and C,p<0.01). Moreover, each combination treatment with desmopressin (1 nMand 1 μM) and 5 nM docetaxel resulted in a further significant reductionin cell migration (FIGS. 2B and C, p<0.01).

Example 3—Anti-Invasive Effects of Desmopressin on PC3 Cells

To assess the anti-metastatic ability of desmopressin, a matrigelinvasion assay was performed. As shown in FIG. 3, 5 nM docetaxel aloneand the two different doses of desmopressin (1 nM and 1 uM) treatmentinduced significantly inhibition of cell invasion. Furthermore, eachcombination treatment of 5 nM docetaxel and desmopressin (1 nM and 1 μM)resulted in a significant reduction in cell invasion (FIG. 3, A and B,p<0.01).

Example 4—Effect of Desmopressin in Combination with Docetaxel on CellCycle

Desmopressin in combination with docetaxel induces cell cycle arrest anddesmopressin enhances the apoptotic effect of docetaxel as determined bywestern blot analysis.

To ascertain the results of MTS assay, a western blot analysis wasperformed to examine cell proliferation. Docetaxel has the ability toalter key regulatory molecules including the suppression of microtubulewith consequent mitotic spindle disruption, leading to G2/M phase cellcycle arrest and induction of bcl-2 phosphorylation ultimately leadingto apoptosis.¹⁶⁻¹⁸ The expression of cyclin A, cyclin B, CDK2 and CDK4were reduced in the cells treated with desmopressin and docetaxel (FIG.4A). In addition the expression of CDK inhibitory protein,p21(waf1/cip1) and p27(kip1) were elevated under the same conditions(FIG. 4A). The results indicate that desmopressin in combination withdocetaxel therapy may inhibit the molecules associated with cell cycleprogression and concomitantly inducing cell cycle arrest. The ratio ofBax/Bcl-2 revealed a 4 fold and 8 fold increased expression in cellstreated with 1 nM desmopressin in combination with docetaxel and 1 μMdesmopressin in combination with docetaxel respectively, all expressedrelative to control (FIG. 4 C). In addition, both combination treatmentsreduced expression of total Caspase 3. Desmopressin treatment alone didnot reduce the expression of bcl-2, PARP or caspase 3 (FIG. 4, B), whichindicates that desmopressin enhances the apoptotic effect of docetaxel.

Example 5—Desmopressin does not Alter Cell Cycle Distribution forTreatment on PC3

Alterations in cell cycle profiles by flow cytometric analysis usingBrdU labeling on cells treated with desmopressin and DTX alone and incombination were examined. Treatment with 5 nM docetaxel or acombination with 1 nM/1 μM desmopressin showed a significant increase inthe proportion of cells in G2 phase consistent with a G2M cell cyclearrest (FIG. 4D). Furthermore, each combination treatment increased thepopulation of cells in the sub G1 phase indicative of apoptosis.

Example 6—Effect of Desmopressin on Migration and Invasion of ProstateCancer Cells and uPA-MMP Pathway Mediates

The two molecules MMPs and uPA are involved in cancer cell invasion,motility and tumor dormancy.¹⁹ Based on the results of migration assayand invasion assay, desmopressin was examined for its anti-metastaticproperties in monotherapy. Dose standardization and time point studieswere carried out with desmopressin. As shown in FIG. 5, desmopressinmonotherapy altered the expression of precursor pro-uPA, active uPA,MMP-2 and MMP-9 all of which were reduced in a dose dependent manner(FIGS. 5A, C and D). In contrast, the expression of uPAR was unaltered(FIG. 5B). In a time point study that was carried out with aconcentration of 1 μM desmopressin, the expression of uPA, MMP-2 andMMP-9 was also reduced in a time dependent manner (FIGS. 6A, C and D).However, uPAR expression was not altered (FIG. 6B). Desmopressinmonotherapy reduced zymogen type uPA, (also called pro-uPA) expression,thus attenuating uPA activity on the cell surface. These resultsdemonstrate that desmopressin has the ability to inhibit tumor cellmigration and invasion via the uPA-MMP pathway (FIG. 7).

It was also demonstrated that desmopressin in combination with docetaxelinhibited tumor cell migration and invasion. As shown in FIG. 6,combination treatment with 5 nM docetaxel and desmopressin (1 nM and 1μM) resulted in a reduction in the expression of uPA when compared tocontrol (FIG. 8, A, p<0.01). In addition, the expression MMP-2 was alsosignificantly reduced in the cells treated with each combination therapycompared to control (FIG. 8 C, p<0.01). The expression of uPAR wasunaltered, as shown in FIG. 8B. The expression of MMP-9 wassignificantly reduced in the cells treated with each combination therapycompared to control (FIG. 8, D, P<0.01). Consequently, desmopressin incombination with docetaxel influences the uPA-MMP pathway.

Example 7—Effect of Desmopressin on PC-3 Cell Growth in a XenograffModel

The effect of desmopressin alone on tumor growth was examined. As afirst step, the inhibition of tumor growth with desmopressin treatmentwas compared to control animals. Using athymic nude mice, cells wereinoculated in matrigel as described above. Tumor volume was assessedtwice weekly. Tumors in control animals grew rapidly measuring a volumeof 923 mm³ on day 35 post tumor inoculation. On the contrary, tumorgrowth in the desmopressin treated mice had a significantly slower rateof tumor development reaching a mean volume of 642 mm3 on day 35 (FIG.9, Student's t-test p<0.01). The tumor volumes between control anddesmopressin treatment were significantly different (ANOVA, p<0.0001).Desmopressin was then examined to determine if it was able to enhancethe sensitivity of PC3 cells to docetaxel in vivo. As shown FIG. 9, thetumors in the combination group were significantly smaller than eithercontrol mice and docetaxel treatment alone (ANOVA, p<0.05). Treatmentwith intravenous injection of docetaxel and/or desmopressin was welltolerated. All mice consistently maintained their body weight duringeach study.

Discussion

Components of the fibrinolytic system urokinase plasminogen activator(uPA) and urokinase plasminogen activator receptor (uPAR) facilitatetargeted proteolysis of the basement matrix in order forneovascularization to occur. Overexpression of these factors in tumortissue has been identified as prognostic for metastatic spread andoverall survival in many human cancers, including hormone dependentcancers such as gastrointestinal, lung, lung, uterine, endometrial,bladder, ovarian, testicular, breast, colon or prostate cancer. Thesefactors are thought to be directly involved in cancer cell invasion andmetastasis. uPA is specifically inhibited by desmopressin.

Desmopressin was found to have anti-proliferative, anti-migration andanti-invasive effects on PC3 cells in vitro and in vivo. Previousreports demonstrated that desmopressin contributes to the reduction oflocolegional disease at the time of primary surgery for advanced mammarycancer¹¹ and inhibited experimental lung colonization when co-injectedintravenously with metastatic mammary tumor cells¹². Ripoll et al.demonstrated that desmopressin had anti-proliferative effects on humancolo-205 and mouse CT-26 colon carcinoma cell lines 20. These studieshave focused on cancer cells expressing the V2 receptor. Desmopressin isa selective agonist for the vasopressin V2 receptor²¹. Typically, the V2receptor is expressed in endothelial cells and the kidney collectingduct, mediating antidiuretic and hemostatic effects. Prostate cancercell lines do not appear to express the V2 receptor²¹⁻²⁴. The Exampleshave demonstrated desmopressin anti-tumor and anti-metastatic effects onPC3 cells lacking the V2 receptor.

The above examples have shown that the anti-metastatic activity ofdesmopressin is mediated through the uPA pathway. Desmopressinmonotherapy resulted in a dose-dependent reduction of uPA expression(FIG. 5A). The uPA and its receptor uPAR are expressed in most solid andinvasive cancers including PC3 cells. The uPA protein involved in thedegradation of the extracellular matrix, facilitating invasiveness andgrowth^(25, 26). The expression of uPA is upregulated in tumor tissue,making it an attractive therapeutic target for cancer therapy^(27, 28).uPA is a member of the serine protease family in strongly implicated asa promotor of tumor progression in various human malignancies includingprostate cancer. It is synthesized and secreted as a pro-enzyme. Bindingto uPAR, uPA efficiently converts the inactive zymogen, plasminogen,into the active seine protease, plasmin. Plasmin can activate MMPs,potent enzyme that can also digest a variety of extracellular matrixcomponents²⁹. The Examples have demonstrated desmopressin as monotherapyand in combination with docetaxel significantly inhibited the expressionof uPA, MMP-2 and MMP-9. It did not alter the expression of uPAR. uPAactivates MMP-2 and MMP-9 during the migration and invasion of prostatecancer³⁰⁻³². The concept of the key role of the binding of uPA to uPARderives from several studies demonstrating that the ability of tumorcells to invade and metastasize is downregulated by uPA inhibitors. Theresults indicate that desmopressin monotherapy reduced pro-uPAexpression as well as active uPA, thus attenuating uPA activity on thecell surface. It has been shown that down-regulation of uPA bydesmopressin inhibits invasion and migration of prostate cancer cells.Thus, without being bound by theory, desmopressin may be acting toinhibit uPA activity in prostate cancer (FIG. 7).

The growth of prostatic tumor cells related to the activation of theplasminogen activator system is derived from the demonstration thatgrowth rates and uPA production in tumor cells cultured at a low densityare higher than those observed in cells grown at higher cell density.This modulation may affect tumor cell proliferation 27. However, theresult of cell proliferation was not consistent with the results of uPAexpression in desmopressin monotherapy. Many cytokines and growthfactors such as TGFβ1, IGF-1, FGF, EGF and bombesin induce theexpression of components of the uPA system^(19, 28, 33, 34). Thesefactors may also be associated with uPA expression and tumor cellproliferation.

Pharmaceutical combinations to reduce dose-limiting toxicity ofdocetaxel and/or to increase its efficacy are attractive. The Exampleshave demonstrated that desmopressin monotherapy inhibited cellproliferation (FIG. 1B) and that combination therapy (desmopressin anddocetaxel) significantly inhibited cell proliferation in vitro and tumorgrowth in vivo compared to control (FIG. 1C, FIG. 9). Desmopressinmonotherapy did not alter cell cycle distribution and the expressionlevels of apoptosis related proteins. Nonetheless, it was observed thatdesmopressin enhanced cell cycle arrest and the apoptotic effect ofdocetaxel in combination.

Desmopressin influences the secretion of a number of factors including,vWF and tPA. vWF is a multimetric plasma glycoprotein that plays animportant role in primary hemostasis, allowing the adhesion of plateletsto the exposed subendothelium³⁵. Terraube V, et al³⁶ reported vWF playsprotective role against tumor cell dissemination in a vWF deficientmutant mouse model. Restoration of vWF plasma levels by administrationof recombinant vWF reduced lung metastasis^(35, 36). Intravenousinjection of desmopressin induces the release of vWF, with a time peaklevels at about 1 hr^(21, 37). It is thought that vWF might be involvedin the interaction of tumor cells with platelets and subendotelium. Onthe other hand, the main function of t-PA is intravascular fibrinolysis.There is a high affinity that t-PA has for fibrin, reflecting highthrombolytic efficacy, since t-PA is synthesized by vascular endothelialcells and secreted into the blood³⁸⁻⁴⁰. In addition, tPA may negativelyregulate angiogenesis and their inhibitors may promote it⁴¹⁻⁴⁶. Hence,vWF and tPA are important factors influencing the impact of desmopressinon tumor cell invasion and metastasis (FIG. 7).

The Examples have shown that desmopressin monotherapy regulated uPA-MMPexpression on PC3 cells at the protein level, not the DNA or RNA levels.Desmopressin alone, or in combination with docetaxel, was shown toreduce tumour volume, inhibit cell proliferation, invasion and migrationof prostate cancer cells. Desmopressin has anti-proliferative,anti-migration and anti-invasive effects in prostate cancer.

Example II

In this study, the anti-tumor effect of desmopressin in combination withdocetaxel using DU145 cells in vitro and in vivo was investigated.

Materials and Methods

Cell Culture

3 Castrate resistant prostate cancer cells DU145 was used. Cell cultureprocedures were followed according to previously described procedures,48, 49.

Chemicals

Docetaxel was purchased from Sigma-Aldrich. Docetaxel was prepared indimethyl sulfoxide (DMSO; Sigma-Aldrich, MO, USA) and diluted with cellculture medium (0.01% DMSO) for cell culture treatments. Desmopressinwas purchased from Ferring© (Octostim 15 μg/mL per ampoule).

Cell Proliferation Assay

Cell proliferation was determined by MTS assay^(48,49). DU145 cells wereplated in 96 well plates at a concentration of 4000 cells/well. After 24hours of attachment, dose standardization was performed, using variousconcentrations of DTX (1 nM, 10 nM, 100 nM and 1 μM) and Desmopressin (1nM, 10 nM, 100 nM and 1 μM each). Cell proliferation was assessed usingMTS assay at 24, 48 and 72 hours following treatment. Based on theresults, cell proliferation assays at similar time points forcombination treatments with DTX 10 nM and 100 nM with Desmopressin 1 μMwere completed. Results were analysed using two-tailed student t-testwith significance level of p<0.05 being considered statisticallysignificant.

Wound Healing Assay

The motility inhibitory potential of desmopressin alone and/or incombination with docetaxel was accessed by wound healing assay accordingto previously described protocol^(59,50). DU145 cell were plated on24-well plates at a concentration of 50,000 cells per well and wereallowed to grow until they reached 90-100% confluence. After incubatingthe cells with 1 mg/L Mitomycin C (Sigma©) for 1 hour, a verticalscratch across each well was created; floating cells removed and cellmedia or media with treatment solutions were added. Images were obtainedat zero-time point and after 24 hours of treatment. A computer-basedmicroscopy imaging system with ×200 magnification (Axiovision©) was usedto measure wound healing of each well. Each experiment was carried outin duplicates and repeated three times

In Vivo Studies with Xenograft Model

All procedures were done according to the Canadian council on animalcare (CCAC) regulations and local animal research ethics boardprocedures and approval.

6-week old male athymic nude mice (Charles river, QC, Canada) were usedto evaluate the effect of combination therapy on DU145 tumor growth invivo. Mice were housed and maintained in laminar flow cabinets underpathogen-free conditions. Following 2 weeks of housing, 1×10⁶ DU145cells per animal in 100 μL matrigel solution (BD Bioscience, CA, USA)were inoculated subcutaneously, according to procedure described byFridman et al (51). After 14 days following inoculation, body weight andtumor size were measured, and mice were randomly assigned to differenttreatment groups. Groups included control (sham treatments), DTX (5mg/kg body weight) intraperitoneally, Desmopressin (2 μg/ml/body weight)intravenously 30 minutes prior to IP injection and 24 hours later orcombination therapy (n=4 per group). Each group received treatment onceevery other week for a total of 3 treatments. Animal weight and tumormeasurements were assessed regularly. Tumor size was calculated bylength (L) and width (W) measured with caliper according to thefollowing formula: V=(L×W2) (n/6).

Two weeks following the last treatment, mice were euthanized; tumorswere excised, measured directly with caliper and sent for histologicalanalysis.

Statistics: Tumor volume calculated during the treatment period werecompared using repeated measures one-way ANOVA test (SPSS©). Final tumorvolume and body weight measurements were compared separately usingone-way ANOVA test.

Results

Cell Proliferation

After determining optimal concentrations of Desmopressin and docetaxel(Data not shown), a combination therapy of 10 nM and 100 nM of DTX and 1μM Desmopressin was used. Combined dosages of 100 nM DTX+1 μMDesmopressin resulted in an inhibition of cell proliferation at 72 hourspost treatment (p<0.05) (FIG. 10). Similar analysis was completed for 24and 48 hours' time points, and with concentrations of 10 nM DTX,revealing minimal response without statistical significance whencompared to DTX treatment alone (Data not shown).

Cell Migration

In vitro wound closure was inhibited by combination treatment comparedto control and each treatment alone for DTX concentration of 10 nMcombined with 1 μM Desmopressin compared to DTX treatment alone (24.8%vs 48.9%, p<0.05, two-way student t-test) (FIG. 11). 10 nM DTX alone hadmigration inhibitory effect when compared to control, while Desmopressintreatment alone had no statistical significant effect. DTX was causingsignificant cell kill at concentration of 100 μM. Although results forthis concentration are shown in FIG. 11, no reliable measurement couldbe drawn and consequently no result could be observed for thoseconcentrations. Representative pictures of in vitro wound closure arepresented in FIG. 12.

In Vivo: Xenograft Mouse Model

Combination therapy (DTX 5 mg/kg I.P and desmopressin 2 μg/ml/bodyweight IV, 30 minutes before chemotherapy and 24 hours later) resultedin decreased tumor volume (FIG. 13), while not effecting changes in bodyweight of the animal (FIG. 14). ANOVA analysis revealed significantdifference in average tumor sizes for combination therapy starting atday 42 post DU145 inoculation, this being the third therapeutic cyclethat was administered to the animals. Representative pictures of themice bearing tumors and the tumors following excision are shown in FIG.15.

Final measurement at day on day 55 post inoculation were done directlyafter tumors were excised, assuming direct measurement would be moreprecise measurements. Results revealed an average tumor size of2049±520, 1597 t 681, 1330 t 550 and 773±314 mm3 for control,Desmopressin alone, DTX alone and combination therapy respectively.(P<0.05 for combination treatment compared to DTX treatment).

Discussion

A combination treatment of DTX and Desmopressin was shown tosignificantly inhibit prostate cancer cells proliferation and migration.These data showed changes in the proliferative and migratory potentialof DU-145 cells as well. In a mouse xenograft model, it was observedthat a combination of Desmopressin and DTX significantly reduced tumorgrowth (as determined by tumor volume) following three treatments ofcombination therapy compared to treatment using single agents.

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1. A method for the treatment of metastatic castrate-resistant prostatecancer comprising: administering to a subject in need thereof atherapeutically effective amount of desmopressin, and a therapeuticallyeffective amount of cabazitaxel.
 2. The method of claim 1, wherein thesubject is a human.